Microreactor, process for the preparation thereof, and process for carrying out a biochemical or biological reaction

ABSTRACT

A microreactor is shown having an inlet or feed channel, an inlet or feed zone for a flow of fluid, a reaction zone, an outlet zone and an outlet or evacuation channel, the zones and channels being in fluid communication, and at least one compound such as an enzyme capable of producing a biological or biochemical reaction with at least one constituent of the flow of fluid, the compound being attached to the surfaces of the inlet zone, reaction zone, and outlet zone.

The present invention relates to a microreactor.

The invention also relates to a process for preparing or manufacturingsuch a microreactor.

Finally, the invention relates to a process for carrying out abiochemical or biological reaction which uses said microreactor.

The field of the invention can be defined as that of miniaturizedsystems of Microsystems which are used essentially for chemical analysisand synthesis.

The favourite fields for chemical microreactors are liquid- andgas-phase reactions, including homogeneous and heterogeneous catalysis,catalytic oxidation, heterocyclic synthesis and photochemical reactions.

In particular, these processes have shown the advantage in usingmicroreactor technology for chemistry in solution and biologicalbioapplications.

Examples of analytical Microsystems or microreactors for synthesis aremore specifically described in documents [1] to [9].

In particular, document [3] concerns the production of nanocolumns forliquid chromatography by micromachining techniques.

The microcolumns comprise “monoliths”, preferably hexagonal monoliths,supporting the stationary phase, and possess inlet and outlet channelshaving a specific architecture with a network of channels, which meansthat the flow of liquid going into the column is divided in two,repeatedly, before it reaches the top of the column.

The total number of channels (C) of the chromatographic column, per se,which can be fed is for example C=2^(n), where n is the number of timesthe liquid flow is divided.

The stationary phase consists of electrostatically bonded poly(styrenesulphate).

The poly(styrene sulphate) is absorbed, from a solution, onto thesurface of the channels after silylation of the walls of the channelsusing (gamma-aminopropyl)trimethoxysilane.

Like document [3], U.S. Pat. No. 6,156,273 describes a column forseparation by chromatography, electrochromatography and electrophoresis,which comprises multiple colocated monolith support structures, whichdefine interconnected channels.

The surfaces of the monoliths can be treated so as to provideinteractions between these surfaces and a sample which crosses theseparation column, in order to perform a separation of the constituentsof the sample.

Among the coatings with which the monoliths can be provided, mention maybe made, for example, of antibody coatings, cationic or anioniccoatings, chelating-agents, organic coatings, such as complex sugars andheparin, gels and reverse-phase coatings, such as C18.

In that document, no reaction takes place in the column, and no newproduct originating from the reaction of the coating which is on thesurfaces of the monoliths with the constituents of the treated sample isgenerated.

That document provides a simple common chromatographic process with moreor less substantial retention of the various constituents of the samplecrossing the column, as a function of their affinity for the coating.

In document [9], since the enzyme and the product to be treated, to bedigested, react in liquid phase, in solution, in the mass of the fluid,the drawbacks of operating in such a way (compared to the invention,where the enzyme is attached to the wall) are as follows: parasiticreactions such as, for example, autolysis in the case of a digestiveenzyme and, consequently, limiting of the concentrations.

The digestion rate can be optimized by confining the digestion reactionsin smaller volumes. The volumes available in integrated devices on chipsmake it possible to use very small amounts of samples, in very smallreaction zones, in order to increase the digestion rates. It hasrecently been shown that it is possible to digest proteins inside wellsproduced in microdevices. As has already been seen, the digestion beingcarried out in solution results in autodigestion of the proteins, whichcan interfere with an analysis by mass spectrometry. This phenomenon isall the more important when it is desired to improve the rate ofdigestion by increasing the concentrations of proteolytic species.

The use of functionalized beads makes it possible to considerably reducethis autolysis, also increases the stability of the enzyme, and provideshigher digestion rates since the amount of digestive enzyme can beconsiderably increased.

However, the operation of filling the microchannels with functionalbeads remains a delicate and relatively unreliable operation.

It emerges from the above text that there is a need for a microreactorwhich makes it possible to carry out chemical and/or biologicalreactions with a very high yield, with a small amount of reactant, suchas an enzyme, and at a rate that is also high.

For example, in the case of enzymatic digestion, there is a need for amicroreactor which, while limiting the problems of autolysis, provideshigh rates and is reliable and easy to use, unlike the microreactorsusing functionalized beads.

The aim of the present invention is therefore to provide a microreactorwhich satisfies, inter alia, these needs.

The aim of the present invention is also to provide a microreactor whichdoes not have the drawbacks, limitations, faults and disadvantages ofthe microreactors of the prior art.

This aim, and even others, are achieved in accordance with the inventionby means of a microreactor comprising an inlet or feed channel, an inletor feed zone for a flow of fluid, an active zone comprising means whichconfer on it a high surface-to-volume ratio, an outlet zone and anoutlet or evacuation channel, said zones and channels being in fluidcommunication, characterized in that the active zone is a reaction zonecomprising at least one compound capable of producing a biological orbiochemical reaction with at least one constituent of said flow offluid, said compound being attached to the surfaces of said reactionzone.

Advantageously, in the microreactor according to the invention:

-   -   said inlet zone comprises means for giving said flow of fluid a        constant flow rate, for evenly distributing the flow of fluid        over the entire cross section of said reaction zone, and for        increasing the surface/volume ratio as the flow of fluid        progresses towards the active zone which is a reaction zone;    -   said at least one compound capable of producing a biological or        biochemical reaction with at least one constituent of said flow        of fluid is attached to the surfaces of said inlet zones, active        zone which is a reaction zone, and outlet zone;    -   said outlet zone comprises means for regrouping the flow of        fluid derived from the active zone which is a reaction zone        comprising the products derived from said biological or        biochemical reaction, and for reducing the surface-to-volume        ratio as the flow of fluid progresses from the active zone which        is a reaction zone towards said outlet channel, and for        evacuating said flow of fluid.

It is clear that the flow of fluid, when it flows in the device of theinvention, encounters successively the inlet zone, then the active zone(reaction zone) and, finally, the outlet zone, then the outlet channel.

The microreactor according to the invention differs, first of all,fundamentally from the device described for example in U.S. Pat. No.6,156,273, which is fundamentally a separation device that obeys theconventional rules of chromatography and not a device aimed at carryingout a reaction, i.e. a reactor.

In this document U.S. Pat. No. 6,156,273, compounds are attached to thewalls of the device, but their aim is to retain to a greater or lesserdegree the various constituents of the liquid sample which crosses thedevice.

This is a typical retention phenomenon in chromatography, the aim ofwhich is to prolong by varying amounts the period of time that theconstituents of the treated liquid spend in the column, in order toensure the staggered exit, and therefore the separation, thereof.

The constituents of the treated liquid undergo no conversion in thecolumn and no new product is generated inside the column. In fact, inthe device of the prior art, the zone which can be defined as an activezone makes it possible to perform chromatography, with no reaction,whereas in the device according to the invention, the active zone is areaction zone since attached therein are compounds capable of producinga reaction.

On the contrary, the invention relates not to a separation device, butto a reactor, which means that the compounds attached to the surfaces ofthe various zones of the device, namely of the microreactor, accordingto the invention, produce a biological or biochemical reaction with atleast one constituent of the flow of fluid, that this constituent isconverted and that new products are created and then collected.

In other words, in the reactor according to the invention, a samplewhich circulates in the reactor will, according to the invention,interact with the compounds capable of reacting, that are attached tothe surfaces of the reactor, and thus create products derived from thisreaction, unlike, again, the device according to document U.S. Pat. No.6,156,273, where the compounds, the coating, attached to the wallssimply promote the more or less prolonged attachment of the constituentsof the sample, but do not generate new products.

The device according to the invention is also fundamentally differentfrom the devices described in particular in document [9], in whichdevices the reaction between the compound capable of producing abiological or biochemical reaction and at least one constituent of theflow of fluid occurs in the liquid phase, in solution, said compoundbeing absolutely not immobilized, attached to the surfaces of thedevice, as in the microreactor according to the invention.

By virtue of this essential characteristic of the microreactor accordingto the invention, the concentrations of the species immobilized on thesurface can be largely increased without the appearance of parasiticreactions such as, for example, autolysis in the case of a digestiveenzyme.

Finally, compared to the microreactors comprising a filling ofmicrobeads, the microreactor according to the invention has theadvantage of being more reliable and simpler to prepare and of having,likewise, a reliable and simple mode of functioning.

The device according to the invention is described as microreactor; thisname is commonly used in this field of the art and is perfectly clear tothose skilled in the art. However, it could be useful to specify thatthe largest dimension of the microreactor according to the invention,which is for example its length or height, is generally from 10 mm to 30mm.

Of course, the applications and/or the nature of the products used mayrequire different dimensions.

The compound capable of producing a biological or biochemical reactionmay be any compound that corresponds to such a definition, but it isgenerally chosen from enzymes.

In other words, it may be any compound capable of interacting with aconstituent, or target molecule, present in the flow of fluid whichcrosses the microreactor, and of converting said constituent by means ofa molecular biology reaction so as to obtain, from this constituent, anew product. It may involve, for example, an interaction and reaction ofthe enzyme/substrate type, where said compound is an enzyme and saidconstituent is a substrate for said enzyme.

Those skilled in the art will find numerous other obvious applicationsof the present invention based on this definition.

According to the invention, when this compound is an enzyme, it may bechosen from the oxidoreductase class, the transferase class, thehydrolase class, the lyase class, the isomerase class, or the ligase orsynthetase class.

It may for example be an enzyme with lytic activity, such as a protease,a nuclease, a lipase, a glycolase, a kinase, etc.; an enzyme having anactivity that modifies or acts on nucleic acids, such as DNA or RNApolymerase, primase, DNA ligase, a nuclease, a reverse transcriptase, akinase, a phosphatase, a phosphorylase, a restriction endonuclease, atopoisomerase, a transferase, etc.

Among proteases, mention may be made, for example, of endopeptidasessuch as pepsin, trypsin, chymotrypsin, cathepsins A, B and C; andexopeptidases such as carbopeptidases, aminopeptidases and dipeptidases.

Preferably, the enzyme is trypsin and the substrate is a peptide or aprotein.

According to the present invention, when the compound which is capableof interacting with the constituent of the fluid is an enzyme, themicroreactor can be called an enzyme microreactor. For example, when theenzyme is trypsin, it may be called a tryptic microreactor; or else, forexample when the enzyme is a polymerase, it may be called a polymerasemicroreactor.

The compound capable of producing a biological or biochemical reactionis attached to said surfaces, for example, by covalent coupling, byinteractions involving ligands, or by any other method for immobilizingthis compound on the surface.

The microreactor according to the invention may have any shape, but itis advantageously substantially elongated in shape, the three zonesdescribed above being defined on a substantially flat substrate, theflow of fluid flowing substantially along the longitudinal axis of saidreactor.

Advantageously, the means of the reaction zone, which confer on it ahigh surface-to-volume ratio, consist of blocks—or monoliths, as theyare denoted in the prior art—comprising a base on said support connectedto a top by means of a wall that is substantially perpendicular to theplane of said substrate, said blocks being evenly spaced out accordingto a two-dimensional network and defining, between their walls, channelsconnected to one another and substantially parallel to the longitudinalaxis of the microreactor and flow axis of the flow of fluid.

Said blocks may have any shape, but advantageously their bases and theirtops will have a shape chosen from discs, ellipses and polygons,preferably regular polygons, such as squares, diamonds, hexagons, etc.

The preferred shape for the base of the blocks or monoliths is that of aregular hexagon or else that of a square.

The size of these blocks is that, for example, of their base and/or top,for example in the form of a square or a regular hexagon, and it isdefined by the fact that this base or top, preferably in the shape of asquare or of a regular hexagon, can fit within a circle having a radiusof 1 to 20 μm, preferably of 2 to 10 μm, for example of 5 μm.

Of course, different dimensions may be required by the applicationsand/or the nature of the products used.

Advantageously, said blocks are arranged in rows, the axis of which issubstantially perpendicular to the longitudinal axis of the microreactoror flow axis of the flow of fluid, the blocks belonging to twosuccessive rows being arranged in staggered rows, i.e. directly shifted.

Advantageously, the spacing between the axes of two successive rows isgenerally 10 to 30 μm, for example 12 μm, and the spacing between thecentres of the bases of two blocks in the same row is 10 to 30 μm, forexample 14 μm.

Of course, different dimensions may be required by the applicationsand/or the nature of the products used.

Advantageously, the means of the inlet zone, defined above, for givingthe flow of fluid a constant flow rate, for evenly distributing the flowof fluid over the entire cross section of the reaction zone, and forincreasing the surface-to-volume ratio as the flow of fluid progressestowards the reaction zone, consist of deflectors comprising a base onthe support connected to a top by means of a wall that is substantiallyperpendicular to the plane of said substrate, said deflectors dividingthe inlet channel into C channels, this division being repeated n times,such that the number of channels at the inlet of the reaction zone isequal to C^(n), n and C being integers, and the total cross section ofthe channels at each division being constant and equal to the crosssection of the inlet channel.

Preferably, C=2 or 3, more preferably C=2 and n is an integer from 2 to10. The only limit for the number n is the selected size of themicroreactor.

Advantageously, the means of the outlet zone for regrouping the flow offluid derived from the reaction zone and for reducing thesurface-to-volume ratio as the flow of fluid progresses from thereaction zone towards said outlet channel—said means being identical tothe means provided in the inlet zone—consist of deflectors comprising abase on the support connected to a top by means of a wall that issubstantially perpendicular to the plane of said substrate, saiddeflectors regrouping the channels of the reaction zone by dividingtheir number by S, this division being repeated m times, so as to form asingle channel or outlet channel. S and m are integers, preferably S=2or 3, more preferably S=2 and m is an integer from 2 to 10. The number mhas the same limits as the number n.

Advantageously, the microreactor also comprises a cover or cap coveringsaid inlet and outlet zones and said inlet and outlet channels.

The cover or cap is optionally provided with inlet and/or outletorifices.

The invention also relates to a set of microreactors, as describedabove, formed on a substrate and comprising from 2 to 50 microreactorsor more depending on the size of each of these microreactors.

Preferably, the microreactors of said set differ from one another bymeans of the shape of the blocks and/or the size of the blocks and/ortheir distribution (for example, space between the blocks and the rows)and/or their length.

The invention also relates to a system formed on a substrate andcomprising at least one microreactor, as defined above, a fluid feedreservoir connected to said inlet or feed channel and a fluid outletreservoir connected to said outlet channel.

According to one embodiment, the fluid feed reservoir is provided on thesubstrate on which the microreactor is formed, for example etched in thesubstrate, and the inlet channel is also provided on the substrate, forexample etched in the substrate.

According to another embodiment, the fluid feed reservoir is placedoutside the substrate on which the microreactor is formed, and the inletchannel—which connects the reservoir to the microreactor—is provided inthe form of a capillary tube.

The same arrangements can be envisaged for the fluid outlet reservoir.

Advantageously, the microreactor, the system or the set, describedabove, may be connected to an analytical device, such as a massspectrometer, preferably by means of a capillary leading to an“electrospray” or to a capillary electrophoresis device.

The invention also relates to a process for preparing a microreactor, asdescribed above, said process comprising the following successive steps:

-   -   etching the three zones of the microreactor and, optionally, the        inlet and outlet channels, in a substantially flat substrate;    -   covering, closing the microreactor by means of a cover or cap;    -   attaching a compound capable of producing a biological or        chemical reactor to the surface of the reaction zone and,        optionally, to the other surfaces of the microreactor.

Preferably, the etching is carried out by a process of isotropic oranisotropic dry etching.

Advantageously, the substrate is made of a material chosen from silica,oxidized silicon, silicon, polymers, plastics and resins, such assilicones, epoxy resins and elastomers.

Advantageously, said attachment is carried out by means of covalentcoupling or by means of interactions involving ligands.

If the substrate is made of silica or of oxidized silicon and thecompound to be attached is an enzyme, then the attachment is carried outby means of the series, sequence, of following steps:

-   -   rehydration in basic medium so as to obtain silanol sites;    -   silanization of the substrate with a reactive epoxy silane, such        as 5,6-epoxyhexyltriethoxy-silane;    -   hydrolysis of the epoxide so as to give a diol;    -   oxidation of the diol to aldehyde;    -   immobilization of the enzyme, such as trypsin, by reaction of        the amine functions of the lysine with the aldehydes;    -   optionally, reaction of the imine bonds thus formed, with a        reducing agent.

Finally, the invention relates to a process for carrying out abiochemical or biological reaction, in which a flow of fluid iscirculated in the microreactor, as described above, in order for atleast one constituent of said flow of fluid to react with the compoundcapable of producing a biological or biochemical reaction, and a flow offluid comprising the product(s) of said reaction is collected at theoutlet of the microreactor.

Preferably, said reaction is a reaction of the enzyme/substrate type,said compound capable of producing a biological or biochemical reactionis an enzyme, said constituent of said flow of fluid is a substrate forthe enzyme, and the product(s) of the reaction is (are) the product(s)derived from the reaction of said enzyme with said substrate.

The enzyme can be chosen from all enzymes, such as they have beendefined above.

Preferably, said reaction is an enzymatic digestion reaction with aprotease, said compound capable of producing a biological or biochemicalreaction is a protease, said constituents of the flow of fluid arepeptides or proteins, and the products of the reaction are peptidesegments.

More preferably, the enzyme is trypsin.

The invention will now be described in detail in the description whichfollows, given by way of nonlimiting illustration, with reference to theattached drawings in which:

FIG. 1 is a sectional side view representing a general diagram of amicroreactor according to the invention;

FIG. 2 is a graph which gives the surface/volume (S/V) ratio (inarbitrary units) for each stage (E) of the inlet of a microreactor,according to the invention;

FIG. 3 is a view from above of the inlet and of the outlet of amicroreactor according to the invention, prepared on a flat substrate;

FIG. 4 is a view from above representing details of the inlet (and ofthe outlet) and of the edge of a microreactor according to theinvention, and showing the arrangement and the designs of blocks of amicroreactor according to the invention, these blocks having a base anda top in the shape of a regular hexagon;

FIGS. 5A to 5F show the production of a microreactor according to theinvention, in a substrate made of silicon, using essentially a processof photolithography;

FIGS. 6 and 7 are photographs, taken on a scanning electron microscope,of a reactor produced according to the invention. In FIG. 6, the scalebar represents 200 μm; and in FIG. 7, the scale bar represents 50 μm.

The microreactor according to the invention, the general diagram ofwhich is illustrated in FIG. 1, comprises an inlet zone (1), a reactionzone (2) forming the “reactor” per se, and, finally, an outlet zone (3).

The microreactor shown diagrammatically in FIG. 1, has an elongatedshape: this is the preferred form of the reactor, with a length, forexample, of 10 to 30 mm, while its small dimension or width or diameteris, for example, from 0.5 mm to 1 mm, which justifies the term“microreactor”. These dimensions are given by way of example and can belargely modified as needed.

It should be noted that the microreactor according to the invention mayhave, for example, the configuration of the column of document U.S. Pat.No. 6,156,273, mentioned above, but it is recalled that it differsfundamentally therefrom by the fact that it comprises a compoundattached to its surfaces allowing a reaction, and that it is therefore areactor and not a separation device.

The inlet zone (1) generally consists of a microchannel, followed by asystem of zigzags arranged in the longitudinal direction of themicroreactor. This system of zigzags or deflectors makes it possible toimpose, for a fixed flow rate of the flow of fluid upstream of themicroreactor, a constant rate of flow throughout the network of channelsthrough the zigzags, before entering the main reactor. The network ofchannels also promotes a homogeneous distribution or dividing-up of thefluid so as to disperse it over the entire width of the reaction zone.

Finally, the dichotomic arrangement, for example, of the zigzags ordeflectors makes it possible to increase the surface/volume ratio as theflow of fluid advances towards the core of the microreactor.

FIG. 2, given by way of example, shows the surface to volume (S/V) ratiofor each stage of the inlet of a microreactor according to theinvention. It is noted that, the closer one gets to the start of thereaction zone (stage 8), the higher the surface/volume ratio.

The increase in the surface-to-volume ratio improves, as it goes along,the yield of the biological or biochemical reaction, until its optimumis reached in the core of the reactor, i.e. in the zone referred to asreaction zone.

The increase in surface is preferably provided by the presence of blocksthat are substantially vertical on a substrate; these blocks may exhibitvarious geometries and also various sizes and various spacings, as isdescribed later, in relation to FIG. 3. The length of the microreactoris also variable.

The length is generally fixed, in order to be able to optimize the yieldof the reactor, for minimum obstruction, dimensions.

The outlet zone of the microreactor (3) is preferably identical in shapeto the inlet; this part makes it possible in particular to regroup thevarious products derived from the biological reaction having taken placein the microreactor, i.e. essentially in the reaction zone (2), beforesubsequently using them, for example analysing them.

It is possible to integrate several microreactors on the same substrate,for example the same wafer, i.e. to form a set of microreactors on thesame substrate.

Preferably, the microreactors that are on the same substrate will bedifferent, the differences relating, for example, to the geometry of theblocks, making it possible to increase the surface-to-volume ratioand/or the spacing between blocks and/or the space between rows and/orthe length of the reactor. The inlet of the reactor is only modifiedaccording to the size of the blocks selected.

FIG. 3 is a view from above of the inlet of the microreactor accordingto the invention, prepared on a flat substrate.

The microreactor is fed by means of a channel referred to as“microchannel” (31) having a width, for example, of 100 μm, which endsin a channel or distributor (32) having a width, for example, of 400 μm,and then, finally, in the first stage (33) of the inlet zone per se,which has a width of, for example, 640 μm.

The channel forming the first stage divides a first time into twochannels (34) and (35), the widths of which are equal, namely: to 320μm, and for which the sum of the widths is equal to that of the singlechannel (33).

In total, the single channel of the first stage is divided six times,each of the channels of a stage being divided into two channels of equalwidth and the total width of the channels of each stage being constantand always equal to the width of the channel of the first stage (640μm). In the final stage of the inlet zone, there are sixty-four channels(36), and each one has a width, for example, of 10 μm.

The dimensions in microns of the microreactor inlet zone, illustrated inFIG. 1, have been indicated in Table I below, these dimensions beinggiven only by way of example:

TABLE I Dimension μm a 1500 b 350 c 300 d 250 e 200 f 150 g 100 h 50 i25 j 100 k 400 l 640 m 320 n 160 o 80 p 40 q 20 r 10 s 128 t 70 u 90 v35 w 15 x 8 y 4 z 2 aa 1 ab 64 ac 32 ad 16 ae 8 af 4

FIG. 3 can represent both the inlet and the outlet of a microreactoraccording to the invention. For this, it is sufficient to reverse FIG.3, the channel (31) then located at the bottom of the figure representsthe outlet or evacuation channel.

FIG. 4 represents the details of the inlet (and of the outlet) and ofthe edge of a microreactor according to the invention.

This figure shows the channels (36), already described above, which endin the reaction zone per se (41) of the microreactor according to theinvention.

In this zone, blocks (45), etc., are arranged in parallel rows (41),(42), (43), (44), etc., and the axis of these rows is perpendicular tothe longitudinal axis of the microreactor and to the flow axis of theflow of fluid.

In FIG. 4, the blocks have a base and a top in the shape of a regularhexagon, each of these hexagons fitting within a circle having a radiusA, which is generally from 1 to 10 μm, for example 5 μm (by way ofexample).

The blocks of two successive rows are arranged in staggered rows andform, with one another, channels that are parallel to the direction offlow 46, which divide into channels 47, which then recombine as channels48 that are once again parallel to the direction of flow.

In the same row, the centres of the blocks, for example of the hexagons,are 10 to 30 μm, for example 14 μm, apart (B) and the axes of twosuccessive rows are generally from 10 to 30 μm, for example 12 μm, apart(C).

In FIG. 4, it is noted that the edge of the reaction zone again takesthe outer shape of the blocks with the aim of maintaining a constantchannel cross section and avoiding dispersion of the rate of flow of thefluid between the edges and the centre of the reactor.

The reaction zone opens out slowly from the inlet zone and the channels(36) by means of a tapered portion defined by the dimensions D and E,for example D=70 μm and E=10 μm, so as to end in the core or centre ofthe reaction zone limited by walls that are essentially parallel, one(50) of which is represented in FIG. 4.

The reaction zone provides virtually all the biological function of themicroreactor.

The increase in surface is precisely provided by the presence of theblocks which can, besides the hexagonal geometry represented in thefigure, have other geometries, for example the blocks may be in the formof a diamond, an ellipse or a disc, and may also have a different sizeand spacing (distance between two blocks), for example of 10 to 30 μm.

Preferably, a cross section that is either hexagonal, as in FIG. 4, orsquare, which fits within a circle of variable diameter, for example of2 to 20 μm, is used, in order to obtain a compromise between a maximumsurface area, defining a network of interconnected microchannels thatare virtually parallel to the longitudinal axis of the microreactor (thepresence of channels perpendicular to the longitudinal axis would induceproduct stagnation and would decrease the yield of the microreactor) andminimizing the complexity of the technological implementation.

As was indicated above, the outlet zone is generally symmetrical to theinlet zone.

The microreactor according to the invention can be produced for anysuitable process, but when it is prepared on or in an essentially flatsubstrate, the microreactor may be, for example, prepared by anisotropic(or isotropic) dry etching in silicon using, for example, a process ofthe ICP-DRIE (inductively coupled plasma, deep reactive ion etching)type.

The units—the term “unit” is intended to mean the feed and evacuationchannels, the inlet, reaction and outlet zones, formed for example bythe deflectors and blocks—are then defined in the silicon by an etchingmask, for example by a photoresist, commonly used in microelectronicsor, for example, by silica, this mask being sufficiently thick to allowthe features to be etched in the silicon, at the thickness chosen by theoperator, for example from 50 μm to 100 μm.

The features can be defined in this etching mask, for example, by alithography process conventionally encountered in microelectronics,followed, for example in the case of silica, by reactive ion etching ofthis material.

The reactors may also be, for example, prepared by anisotropic dryetching in silica, it being possible for the selected protective mask tothen be, for example silicon. The microreactor may also be prepared inother materials, for example made of polymers, such as epoxy resins,elastomers, plastics.

The use of microtechnology makes it possible to produce, by means ofanisotropic or isotropic etching, structures that have complexgeometries and very large surface-to-volume ratios, without thedrawbacks of microbead fillings.

The reactor can then be covered, for example with a PDMS(polydimethylsiloxane) sheet, which may or may not comprise inlet and/oroutlet orifices, after treatment of said cover and of the reactor withan oxygen plasma, as described in the literature. In this case, PDMS isknown to have properties of spontaneous adhesion on most solid supports.

The reactor may also, for example, be covered by molecular bonding of asilica plate or of a glass plate, which may or may not comprise inletand/or outlet orifices, after cleaning and chemical preparation of thetwo hydroxylated substrates (substrate SiO₂ on silicon/glass or silicacover). The presence of silanol sites (SiOH) at the surfacespontaneously attracts water molecules, and the two parts of themicrocomponent, namely: cover and reactor, bond to one another via watermolecules. Some of the water contained between the two surfaces iseliminated by heating, until about three layers of water molecules whichmake the adhesion possible are obtained.

Alternatively, the microreactor may be, for example, covered by anodicbonding of a glass plate, which may or may not comprise inlet and/oroutlet orifices.

Alternatively, the microreactor may, for example, be covered by bondingof a polymer plate chosen by the user, which may or may not compromiseinlet and/or outlet orifices, using for example a screen-printingadhesive coating process.

This type of bonding consists of three main steps: screen printing,which consists in applying adhesive only to certain zones of thesubstrate, bonding, which consists in bringing the substrate locallycoated with adhesive into contact with the cover and, finally, heating,which induces polymerization of the adhesive. The curing can be carriedout photochemically if the adhesive is UV-polymerizable.

Finally, the microreactor may be, for example, covered by SDB (silicondirect bonding) to a silicon plate, which may or may not comprise inletand/or outlet orifices.

The attachment of the constituent having the biological or biochemicalfunction in the microreactor, which may be for example an enzyme of thetrypsin type, can be carried out according to various methods:

-   -   by means of covalent coupling linking the molecule to be        attached to the surface of the microreactor;    -   by means of interactions involving ligands.

Thus, in the case of an enzyme such as trypsin, the attachment thereofto a silica substrate can be carried out by means of successive steps ofrehydration, silanization, for example with a reactive epoxy silane,hydrolysis, oxidation and, finally, immobilization, attachment of theenzyme via —NH₂ bonds carried by the lysine groups of the trypsin.

Scheme 1 below illustrates the steps and the operating conditions whichcan be used to immobilize an enzyme such as trypsin.

By way of indication, trypsin carrying a biotin function (biotinylatedtrypsin, type XI-B from SIGMA ALDRICH®) was attached to circularsurfaces of the order of 1 mm in diameter.

After a step consisting in labelling with streptavidin carrying a Cy₃fluorophore, and excitation at 550 nm, a fluorescence image is obtainedat 580 nm, the intensity of which is 225 AU (arbitrary units). Thesignal/noise (trypsin/substrate) ratio is between 35 and 40.

To carry out a reaction inside the microreactor according to theinvention, the flow of liquid of which one of the constituents can reactwith the compound attached to the walls of the microreactor is passedthrough using an appropriate device, such as a syringe pump and itsassociated syringe, or the like. The flow rate is preferably a constantflow rate, which ensures that the amount of time spent inside thereactor is from 1 to 15 minutes, for example as a function of thekinetics of the reaction in the microreactor. The products of thereaction are sent towards an analytical device, such as a massspectrometer, or towards another use, for example a capillaryelectrophoresis.

The invention will now be described with reference to the followingexamples, given by way of non-limiting illustration.

EXAMPLES Example 1

This example illustrates the production of a silicon microreactor, withreference to the attached FIG. 5.

A layer of photoresist (52) of the SHIPLEY® S 1813 type is deposited byspin-coating onto a four-inch (10.16 cm) silicon substrate (51) of <100>type and of thickness 525 μm.

Lithography is then performed by means of a UV exposing beam (53)through a mask (54) provided with or having n features defining thegeometry of the microreactors; the exposure time is 10 seconds.

This pattern is subjected to deoxidation of the pattern bottoms by meansof a Nextral NE 110 RIE device in a CHF₃/O₂ atmosphere at a flow rateratio of 50/10 sccm, under a pressure of 100 mT, with a power of 10 W,for 1 minute.

Next, the zones not protected by the resin are etched using a deepetching device of the Multiple STS ICI DRIE type.

The following step consists in cutting the mask of resin with fumingHNO₃ without ultrasound for 5 minutes.

The sidewalls of the etched patterns are then cleaned by oxidation in atube furnace in oxygen for 50 minutes at 1000° C. and chemicaldeoxidation with HF for a few seconds.

Thick oxidation of the patterns over a thickness of 3 μm (55) is thencarried out in a tube furnace under steam at 1000° C. for 18 hours and50 minutes.

Each microreactor is then cut out and separated from the wafer. Apolydimethylsiloxane cover is then used to close the microreactor. Thebonding of the cover and of the microreactor is carried out after oxygenplasma treatment of the two surfaces to be brought into contact (TEGALO₂ plasma device, pressure 100 mTorr, activation time 30 seconds). Themicroreactor is connected with any device for circulating fluid, viacapillaries inserted into the inlet and outlet channels of themicroreactor.

The microreactor used in the digestion example has a hexagonal blockgeometry with blocks that are 10 microns in diameter, separated from oneanother by 14 microns along an axis perpendicular to the direction ofthe flow of liquid, and by 12 microns along an axis parallel to the flowof the liquid. The depth of the microreactor, defined as being theaverage height of the blocks, is 50 microns.

Example 2

In this example, trypsin is attached, immobilized on the surfaces of themicroreactor produced in Example 1, so as to obtain a “functionalized”microreactor according to the invention.

The immobilization of the trypsin is then verified by fluorescence.

The trypsin used is a type I trypsin from bovine pancreas, sold by thecompany SIGMA ALORICA® (ref. T 8003).

The reaction mechanism is composed of several steps, namely:

-   -   rehydration in basic medium making it possible to obtain silanol        sites, silanization of the substrate with        5,6-epoxyhexyltriethoxysilane resulting in the formation of        films;    -   hydrolysis so as to convert the epoxide function into a diol        function;    -   oxidation to an aldehyde function and, finally, immobilization        of the enzyme by reaction of the amine functions carried by the        lysines, with the aldehyde functions. The imine bonds thus        formed are stabilized by reaction with a reducing agent. The        precise protocol used in this example is described below.        1. Rehydration

NaOH Brown: NaO 4.9 g; EDI 14.7 ml; EtOH 19.6 ml;filling of the microreactor at 3 μl/m, reaction for 2 hours at ambienttemperature. Emptying of the reactor under a stream of nitrogen, thenwashing with deionized water (100 μl at 3 μl/min). Oven for 15 minutesat 80° C. Rinsing with toluene (50 μl at 3 μl/min).2. Silanization

-   Toluene 10 ml;-   DIEA 50 μl;-   5,6-Epoxyhexyltriethoxysilane 25 μl;-   filling of the microreactor at 3 μl/min, and then washing with    ethanol (100 μl at 3 μl/min);-   crosslinking for 3 hours at 110° C.    3. Hydrolysis

0.2N HCl. Filling of the reactor at 3 μl/min. Reaction under a stream ofHCl for 3 hours at ambient temperature. Emptying, then washing withdeionized water (100 μl at 3 μl/min).

Drying for 30 minutes at 110° C.

4. Oxidation

-   NaIO₄ 6.6 μg;-   Deionized water 3 ml.

Reaction under a stream of said reactants (100 μl at 3 μl) for 1 hour atambient temperature. Emptying and drying under a stream of nitrogen.

5. Immobilization of the Enzyme

The solution containing the enzyme to be immobilized is introduced intothe microreactor at a fixed flow rate. Once the microreactor is full,its ends are blocked off with parafilm. The immobilization reaction isthen carried out in static mode.

Using this reaction scheme and the associated protocol, biotinylatedtrypsin (biotinylated trypsin type XI-B from SIGMA ALDRICH® provided inthe form of a solution of 2.5 mg/ml of trypsin, 0.1M Na₂HPO₄, 0.05 MNaCNBH₃) was immobilized on the internal surface of the microreactorexhibiting columns 10 μm in diameter, 5 μm apart, and equal to 50 μm inheight, and then visualised with a solution of streptavidin Cy₃.

The biotin/streptavidin-Cy₃ couple is visualised by fluorescence at 570nm.

Observation under an epifluorescent microscrope makes it possible tovisualise the presence of trypsin over the entire surface of themicrocolumns and over the entire tryptic reactor.

Example 4

In this example, digestion of BSA is carried out in the microreactorprovided with an immobilized enzyme, according to the invention,prepared in Example 3.

By means of a syringe pump, the inlet reservoir of the microreactor isfilled with a solution of bovine serum albumin (BSA) at 2 mg/ml with0.05% of NaN₃, at an average flow rate of the order of 5 μl/min. Theamount of time spent in the microreactor by the protein to be digestedis of the order of 5 minutes. After a few minutes, the volume infusedthrough the microreactor is sufficiently large to allow correct analysison a MALDI_TOF mass spectrometer. The spectrum obtained shows peptidesegments derived from the digestion of the BSA.

REFERENCES

-   [1] P. D. I. FLETCHER, S. J. HASWELL, V. N. PAUNOV, Analyst 124,    1273-1282 (1999)-   [2] T. McCREEDY, Analytica Chimica Acta 427, 39-43 (2001)-   [3] B. He, H. TAIT, F. REGNIER, Analytical Chemistry 70, 3790-3797    (1998)-   [4] B. He, L. TAN, F. REGNIER, Analytical Chemistry 71, 1464-1468    (1999)-   [5] B. He, J. Ji, F. REGNIER, Journal of Chromatography A 853,    257-262 (1999)-   [6] B. He, B. J. BURKE, X. ZHANG, R. ZHANG, F. REGNIER (Website of    Analytical Chemistry, 2000)-   [7] B. E. SLENTZ, N. A. PENNER, F. REGNIER, Journal of    Chromatography A, to be published not yet communicated (2001)-   [8] L. XIONG, F. REGNIER, Journal of Chromatography A 924, 165-176    (2001)-   [9] Chemical Abstracts 2001: 335 569, MIYAZAKI M. et al. Chem. Lett.    (2001), (5), 442-443-   [10] Chemical Abstracts 2000: 811 299 CAPLUS

1. A microreactor substantially elongated in shape and formed on asubstantially flat substrate comprising: (i) an inlet or feed channelhaving deflectors dividing the inlet channel into 2 to 10 channels, (ii)an inlet or feed zone for a flow of fluid, (iii) an active zonecomprising means which confer on it a high surface-to-volume ratioconsisting of blocks comprising a base on a support connected to a topvia a wall that is substantially perpendicular to the plane of saidsubstrate, said blocks being evenly spaced out by 10 to 30 μm betweenthe axes of two successive rows according to a two-dimensional networkand defining, between the walls, channels connected to one another andsubstantially parallel to the longitudinal axis of the microreactor andflow axis, and said blocks having a base or top that can fit within acircle of 1 to 20 μm, (iv) an outlet zone and an outlet or evacuationchannel having deflectors dividing the outlet channel into 2 to 10channels, said zones and channels being in fluid communication in whichthe flow of fluid is substantially along the longitudinal axis of saidreactor, wherein the active zone is a reaction zone comprising at leastone enzyme capable of producing a biological or biochemical reactionwith at least one substrate for said enzyme, said at least one enzymebeing attached to the surfaces of said reaction zone whereby said atleast one enzyme reacts with said at least one substrate to create a newcompound having a different chemical structure as a reaction product,wherein said reaction product may be obtained at the outlet channel ofthe microreactor, and wherein said enzyme is chosen from theoxidoreductase class, the lyase class, the isomerase class, or theligase or synthetase class.
 2. A microreactor according to claim 1,wherein: said inlet zone comprises means for giving said flow of fluid aconstant flow rate, for evenly distributing the flow of fluid over theentire cross section of said reaction zone, and for increasing thesurface/volume ratio as the flow of fluid progresses towards the activezone which is a reaction zone said at least one enzyme capable ofproducing a biological or biochemical reaction with at least onesubstrate for said enzyme is attached to the surfaces of said inletzones, active zone which is a reaction zone, and outlet zone; saidoutlet zone comprises means for regrouping the flow of fluid derivedfrom the active zone which is a reaction zone comprising the productsderived from said biological or biochemical reaction, and for reducingthe surface-to-volume ratio as the flow of fluid progresses from theactive zone which is a reaction zone towards said outlet channel, andfor evacuating said flow of fluid.
 3. A microreactor according to claim1, wherein the largest dimension of said microreactor is from 10 mm to30 mm.
 4. A microreactor according to claim 1 wherein the enzyme is anenzyme with lytic activity, or an enzyme having an activity thatmodifies or acts on nucleic acids.
 5. A microreactor according to claim1, wherein the protease is chosen from endopeptidases and exopeptidases.6. A microreactor according to claim 1, wherein the enzyme is trypsinand the substrate for this enzyme is a peptide or a protein.
 7. Amicroreactor according to claim 1, wherein the compound capable ofproducing a biological or biochemical reaction is attached to saidsurfaces by means of covalent coupling or by means of interactionsinvolving ligands.
 8. A microreactor according to claim 1, wherein thebase and the top of said blocks have a shape chosen from ellipses, discsand polygons.
 9. A microreactor according to claim 1, wherein the blockshave a base and a top in the shape of a regular hexagon.
 10. Amicroreactor according to claim 1, wherein the blocks have a base in theform of a square.
 11. A microreactor according to claim 1, wherein thebase and the top of said blocks can fit within circles having a radiusof 1 to 20 μm.
 12. A microreactor according to claim 1, wherein saidblocks are arranged in rows, the axis of which is substantiallyperpendicular to the longitudinal axis of the microreactor or flow axis,the blocks belonging to two successive rows being arranged in staggeredrows.
 13. A microreactor according to claim 12, wherein the spacingbetween the axes of two successive rows is 10 to 30 μm, and the spacingbetween the centers of the bases of two blocks in the same row is 10 to30 μm.
 14. A microreactor according to claim 1, wherein the means of theinlet zone for giving the flow of fluid a constant flow rate, for evenlydistributing the flow of fluid over the entire cross section of thereaction zone, and for increasing the surface-to-volume ratio as theflow of fluid progresses towards the reaction zone, consist ofdeflectors comprising a base on the support connected to a top by meansof a wall that is substantially perpendicular to the plane of saidsubstrate, said deflectors dividing the inlet channel into C channels,this division being repeated n times, such that the number of channelsat the inlet of the reaction zone is equal to C^(n), n and C beingintegers, and the total cross section of the channels at each divisionbeing constant and equal to the cross section of the inlet channel. 15.A microreactor according to claim 14, wherein C=2 or 3 and n is aninteger from 2 to
 10. 16. A microreactor according to claim 1, whereinthe means of the outlet zone for regrouping the flow of fluid derivedfrom the reaction zone and for reducing the surface-to-volume ratio asthe flow of fluid progresses from the reaction zone towards said outletchannel, said means being identical to the means provided in the inletzone, consist of deflectors comprising a base on the support connectedto a top by means of a wall that is substantially perpendicular to theplane of said substrate, said deflectors regrouping the channels of thereaction zone by dividing their number by S, this division beingrepeated m times, so as to form a single channel or outlet channel. 17.A microreactor according to claim 1, also comprising a cap or covercovering said zones and said channels, optionally provided with inletand/or outlet orifices.
 18. A set of microreactors according to claim 1,formed on a substrate and comprising from 2 to 50 microreactors.
 19. Aset of microreactors, said microreactors according to claim 18 whereinsaid microreactors differ from one another by means of the shape of theblocks and/or the size of the blocks and/or their distribution and/orthe length of the microreactor.
 20. A system formed on a substrate andcomprising at least one microreactor according to claim 1, a fluid feedreservoir connected to said inlet or feed channel, and a fluid outletreservoir connected to said outlet channel.
 21. A system according toclaim 20, wherein the fluid feed reservoir is provided on the substrateon which the microreactor is formed, and the inlet channel is alsoprovided on the substrate.
 22. A system according to claim 20, whereinthe fluid feed reservoir is placed outside the substrate on which themicroreactor is formed, and the inlet channel is provided in the form ofa capillary tube.
 23. A process for preparing a microreactor accordingto claim 1, said process comprising the following successive steps:etching the three zones of the microreactor and, optionally, the inletand outlet channels, in a substantially flat substrate; covering,closing the microreactor by means of a cover or cap; attaching acompound capable of producing a biological or chemical reactor to thesurfaces of the reaction zone and, optionally, to the other surfaces ofthe microreactor.
 24. A process according to claim 23, wherein theetching is carried out by a process of isotropic or anisotropic dryetching.
 25. A process according to claim 23, wherein the substrate ismade of a material chosen from silica, silicon, oxidized silicon,polymers, plastics and resins.
 26. A process according to claim 23,wherein said attachment is carried out by means of covalent coupling orby means of interactions involving ligands.
 27. A process according toclaim 23, wherein the substrate is made of silica or of oxidized siliconand the compound to be attached is an enzyme, and the attachment iscarried out by means of the following succession of steps: rehydrationin basic medium so as to obtain silanol sites; silanization of thesubstrate with a reactive epoxy silane; hydrolysis of the epoxide so asto give a diol; oxidation of the diol to aldehyde; immobilization of theenzyme by reaction of the amine functions with the aldehydes;optionally, reaction of the imine bonds thus formed, with a reducingagent.
 28. A process for carrying out a biochemical or biologicalreaction, wherein a flow of fluid is circulated in a microreactoraccording to claim 1, in order for at least one substrate to react withthe enzyme capable of producing a biological or biochemical reaction,and a flow of fluid comprising the product(s) of said reaction iscollected at the outlet of the microreactor.
 29. A process according toclaim 28, wherein said reaction is an enzymatic digestion reaction witha protease, said enzyme capable of producing a biological or biochemicalreaction is a protease, and said substrate is a peptide or a protein,and the products of the reaction are peptide segments.
 30. A processaccording to claim 29, wherein the enzyme is trypsin.
 31. A microreactoraccording to claim 4, wherein the enzyme with lytic activity is aprotease, a nuclease, a lipase, a glycolase, or a kinase.
 32. Amicroreactor according to claim 4, wherein the enzyme having an activitythat modifies or acts on nucleic acids is DNA or RNA polymerase,primase, DNA ligase, a nuclease, a reverse transcriptase, a kinase, aphosphatase, a phosphorylase, a restriction endonuclease, atopoisomerase or a transferase.
 33. A microreactor according to claim 5,wherein the endopeptidases are chosen from pepsin, trypsin,chymotrypsin, cathepsin A, cathepsin B and cathepsin C, and theexopeptidases are chosen from carbopeptidases, aminopeptidases anddipeptidases.
 34. A microreactor according to claim 8, wherein the baseand the top of said blocks have a shape chosen from regular polygons.35. A microreactor according to claim 34, wherein the regular polygonsare diamonds, squares or hexagons.
 36. A microreactor according to claim11, wherein the base and the top of said blocks are in the form ofsquares or of regular hexagons.
 37. A microreactor according to claim11, wherein the base and the top of said blocks can fit within circleshaving a radius of 2 to 10 μm.
 38. A process according to claim 23,wherein the substrate is made of a material chosen from silicones, epoxyresins and elastomers.
 39. A microreactor according to claim 1, whereinthe compound capable of producing a biological or biochemical reactionis attached to said surfaces by means of covalent coupling.